Purpose: :
Mutations in the Retinitis Pigmentosa GTPase Regulator (RPGR) gene cause approximately 15% of retinitis pigmentosa (RP). All RPGR protein isoforms include an N-terminal RCC1-like domain (RLD); however, a majority of human mutations are reported in an unusual, alternately spliced form, RPGR-ORF15, which is highly expressed in photoreceptor (PR) connecting cilium. Though RPGR is reported to be a guanine nucleotide exchange factor for Rab8 or another GTPase, the precise role of RPGR isoforms in PR function and RP-pathogenesis is not understood. Clinical studies have suggested that N-terminal mutations may have faster rates of disease progression. We have therefore constructed a knock-in, mouse model of the syndromic, human RPGR G173R, RLD mutation to evaluate disease pathology and to compare with the existing Rpgr knock-out mouse and Rd9, a mouse that harbors an ORF15 mutation.

Methods: :
A knock-in construct designed to introduce the change of G173R into mouse Rpgr locus was constructed from a BAC clone through ET-recombination. This construct was electroporated into R1 ES cells, and homologous recombination events were selected by genomic Southern. Germline transmitted F1 mice were confirmed for the presence of the knockin allele by long range genomic PCR and served as founders. The hemizygous male mice carrying G173R mutation were analyzed over a 6-month period for RPGR mRNA and protein expression, ERG and photopigment localization.

Results: :
In the knock-in mouse, RPGR transcripts levels for all splice forms were similar to wild type mice, but both RPGR-ORF15 and RPGR 1-19 (constitutive form) proteins showed remarkable reduction. Photopic b-wave was modestly reduced by 2 months but the scotopic a- and b-wave amplitudes were indistinguishable from wild type litter-mates at 6 months. M-opsin was mislocalized but rhodopsin localization remained normal.

Conclusions: :
At 6 months of age, the Rpgr G173R knock-in model shows a profound reduction in both forms of RPGR protein, suggesting that this missense allele confers instability to the RPGR protein. Consistent with an insufficiency of RPGR, retinal function declines over time. Further investigations are in progress to elucidate the impact of Rpgr missense mutation(s) on retinal disease phenotype.